When 1st-round PCR amplification was finished , I could get a clear expected band, but the amount was very low, so I cut the band and run second-round PCR with same condition, it didn't work, I tried so many condition (changed annealing temperature, different concentrations of primer, add GC enhancer), it still didn't work, why?
Dear Aiguo,
Are you dealing with Nested PCR? Also, how you are using the DNA band for PCR? I mean as such band or elution. In my knowledge, here many factors (e.g. method used to elute the nucleic acid from band, conc. of nucleic acid obtained from elution etc.) can influence your results. I would suggest you to once try with various dilutions (1:5, 1:10, 1:20 and 1:50) of template.
In what way did it not work....no signal at all or a long smear down the gel in each lane. Ordinary agarose can inhibit pcr if you just ran crude band extract but the most likely fail is due to over amplification. 10cycles of pcr is 1000 times more product and 20 cycles is 1,000,000 time more product. At these concentrations the product self anneals and generates smears of long pcr product of random length.
I would either use nested primers for the second round pcr or
dilute the band 1:1000 and set up 8 tubes of pcr if you are using the original primers again. Set up the pcr for 1ul of diluted product and 26 cycles and remove tubes at 12,14,16,18,20,22
24 and 26 cycles. A couple of these cycle numbers will give a good yield of product before over amplification begins and the smear appears
Thanks @Rajeev Meora a lot, I cut the band and recovered the DNA with gel extraction kit, and checked the eluded DNA from gel, also I diluted the template 10X, 100X, 1000X, it can't amplify the PCR product with smear band@ Paul Rutland , also I checked the PCR prpduct after 2, 4, 8, 16 cycles, no clear bands thanks.
Aiguo Xu That is unexpected. I wonder if:
1. one primer is degrading so the first pcr is poor but the second is even worse.It sometimes happens to primers dissolved in water not TE
2 It is rare but sometimes one primer alone can produce a pcr product. In this case the increase in product amount may not be exponential but linear so a poor yield on reamplification and nothing to see. Was the first band a really good size match for the expected size?
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